The goal of achieving longstanding site- specific tolerance or donor-specific unresponsiveness to allogeneic tissue without the need for combined systemic immune-suppression is of clinical importance in transplantation medicine in view of the mortality and morbidity associated with rejection of an allograft. To date therapeutic control over both acute and chronic graft rejection has been achieved predominantly with immunosuppressive agents targeted against the function of activated recipient alloantigen-specific T cells. The clinical benefit of these systemic immunosuppressive therapies is achieved at the cost of an increased incidence of life threatening opportunistic infections and transplantation associated malignancies. Novel immunosuppressive agents have been developed which attempt to interfere with rejection by interrupting molecular events involved in the direct and indirect recognition of alloantigens and thereby inducing tolerance using the combination of pretreatment of the recipient with donor antigen and Continued 1 1 R21 AI49858-01 3 SEP Poznansky, Mark C. a short course of immune suppressive or immunomodulatory therapy. Other novel targets for the induction of donor-specific unresponsiveness to allogeneic tissue have been proposed and include the suppression of the trafficking or migration of recipient immune cells into the allograft. We have recently identified a novel biological mechanism, which we have termed chemofugetaxis, and which is defined as the active migration of immune cells away from a chemokine. In particular, we have identified several chemokinetic factors, including the chemokine, stromal-cell derived factor-1 (SDF-1), which elicit a chemofugetactic response from human T cells in vitro and abrogate T-cell migration into a specific anatomic site in response to an antigen challenge in a murine model in vivo. In addition, we have demonstrated that a high level of SDF-1 generated by human bone marrow and thymus stroma contributes to the exclusion of mature T cells from these anatomical spaces in vivo. We hypothesize that the constitutive expression of a chemokine that elicits a chemofugetactic response from T cells by allogeneic transplanted tissue could lead to the exclusion of recipient immune effector cells from the allograft. This would create an immune privileged site for the allograft and represent an entirely novel method by which longstanding site-specific tolerance could be induced and graft rejection prevented in vivo. The application has two specific aims: 1) The identification of CXC- and CC-type chemokines that elicit a chemofugetactic response from activated T cells. The migratory responses of subpopulations of T cells relevant to graft rejection in vivo would be quantitated using a battery of in vitro transmigration assays. 2) The development of a murine model to quantitate the effectiveness of the expression of a chemokine that elicits a chemofugetactic response from activated recipient T cells in preventing or abrogating graft rejection in vivo. This application represents the first step toward the use of chemofugetactic agents in a novel therapeutic approach to induce longstanding site-specific functional tolerance by causing the long-term reversal of the migration of alloantigen-specific recipient immune effector cells into transplanted allogeneic organs. The achievement of the goals presented herein could ultimately contribute to the prevention of acute and chronic graft rejection and the minimization of the need for concomitant treatment of transplant recipients with systemic immunosuppressive agents.